Biosensor

ABSTRACT

There is provided a cholesterol sensor with high-accuracy and excellent response, whose object to be measured is whole blood, where plasma with  which is obtained by filtering out hemocytes therein filtered  in blood can rapidly reach an electrode system. In a biosensor where plasma with  which is obtained by filtering out hemocytes therein filtered with  by a filter is sucked into a sample solution supply pathway due to capillarity, there are formed: a first pressing part for holding a primary side portion of the filter from the bottom; a second pressing part for holding a secondary side portion of the filter from the top and the bottom; a third pressing part for holding the central portion of the filter from the top; and a void for surrounding the filter between the second pressing part and third pressing part.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Section 371 of International Patent ApplicationNo. PCT/JP02/04826, filed May 17, 2002, the disclosure of which isincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a biosensor, specifically a cholesterolsensor, capable of carrying out speedy, highly-sensitive, simpledetermination of a specific component in a sample.

BACKGROUND ART

A description will be given to an example of a conventional biosensor interms of a glucose sensor.

A typical glucose sensor is obtained by forming an electrode systemincluding at least a measurement electrode and a counter electrode on aninsulating base plate by a method such as screen printing and thenforming an enzyme reaction layer including a hydrophilic polymer,oxidoreductase and an electron mediator on the electrode system. Asoxidoreductase used is glucose oxidase; as the electron mediator used isa small complex, an organic compound or the like, such as potassiumferricyanide, ferrocene derivative or quinone derivative. A buffer isadded to the enzyme reaction layer as required.

When a sample solution containing a substrate is added dropwise onto theenzyme reaction layer in this biosensor, the enzyme reaction layerdissolves to cause a reaction of the enzyme with the substrate, whichaccompanies reduction of the electron mediator. After completion of theenzyme reaction, the substrate concentration in the sample solution canbe determined from a value of oxidation current, which is obtained whenthe reduced electron mediator is electrochemically oxidized.

In this type of glucose sensor, a reductant of the electron mediatorgenerated as a result of the enzyme reaction is oxidized at theelectrode, to determine the glucose concentration from the oxidationcurrent value.

Such a biosensor is theoretically capable of measuring diversesubstances by using an enzyme whose substrate is an object to bemeasured. For example, when cholesterol oxidase or cholesteroldehydrogenase is used as oxidoreductase, it is possible to measure acholesterol value in a serum to be used as a diagnostic indicator invarious medical institutions.

Because the enzyme reaction of cholesterol esterase proceeds veryslowly, with an appropriate surfactant added thereto, activity ofcholesterol esterase can be improved to reduce the time required for theoverall reaction.

However, the surfactant, as being included in the reaction system, hasan adverse effect on hemocytes, making it impossible to measure wholeblood itself, as done in the glucose sensor.

Thereat, a proposal has been made to provide a filter(hemocyte-filtering out portion) in the vicinity of an opening of asample solution supply pathway for rapid supply of only plasma withwhich is obtained by filtering out hemocytes therein filtered in bloodinto a sensor. When the filter is inappropriately built in the sensor,however, the hemocytes captured in the filter are destroyed andhemoglobin dissolves out.

As hemocyte components get smaller to about the size of the hemoglobin,filtration of filtering out the hemocyte components with the filterbecomes difficult, whereby the hemoglobin flows into the sample solutionsupply pathway to cause a measurement error.

This is presumably caused by the fact that a difference in thicknessbetween the filter before absorbing a sample solution and the filterexpanded after absorbing the sample solution is not fitted to a gapbetween pressing parts for holding the filter from the top and bottom.When the gap between the pressing parts for holding the filter from thetop and the bottom is too narrow for the thickness of the filterexpanded, the filter is prevented from expanding. The pore size of thefilter thus prevented from expanding cannot widen sufficiently,destroying the hemocytes as infiltrating thereinto.

As opposed to this, when the gap between the upper and lower pressingparts is previously set wide for the supposed thickness of the expandedfilter, it is feared that the filter may be sliding during storage sinceeach sample solution has a different hematocrit value (ratio of red cellvolume), resulted from which degrees of expansion of the filter alsodiffer, depending on sample solutions.

Moreover, when a filter is made thinner than a conventional one in orderto reduce the amount of a sample solution, mere suction of the samplesolution from the termination of a primary side portion of the filter,like a conventional method (Japanese Patent Application No.2000-399056), reduces the amount of the sample solution that can beabsorbed within a certain period of time. For this reason, plasma flowsout of a secondary side portion of the filter at a slower rate, and theinside of a sensor, especially the inside of a sample solution supplypathway, is saturated with the plasma at a slower rate, resulting inlonger measurement time.

As opposed to this, when a suction area is made wider for increasing theamount of the sample solution that can be absorbed within a certainperiod of time and then the sample solution is dropped thereonto fromthe upper part of the filter, the sample solution flows along thesurface of the filter at a faster rate than it infiltrates into thefilter. The sample solution having flown along the surface of the filterthen flows into the sample solution supply pathway from the openingthereof connecting the sample solution supply pathway to the filter,which may lead to a measurement error.

It is an object of the present invention to provide a biosensor improvedsuch that plasma with which is obtained by filtering out hemocytestherein filtered in blood reaches an electrode system with rapidity inorder to obviate the disadvantages thus described.

It is an object of the present invention to provide a cholesterol sensorwith high-accuracy and excellent response, whose object to be measuredis whole blood.

DISCLOSURE OF INVENTION

A biosensor of the present invention comprises: an insulating baseplate; an electrode system having a working electrode and a counterelectrode which are provided on the base plate; a reagent including atleast oxidoreductase and an electron mediator; a sample solution supplypathway which includes the electrode system, and the reagent and has anair aperture on the termination side thereof; a sample supply part; anda filter which is disposed between the sample solution supply pathwayand the sample supply part and which filters out hemocytes, where plasmawith which is obtained by filtering out hemocytes therein filtered within blood by the filter is sucked into the sample solution supply pathwaydue to capillarity, and is characterized by further comprising: a firstpressing part for holding a primary side portion of the filter from thebottom; a second pressing part for holding a secondary side portion ofthe filter from the top and the bottom; a third pressing part forholding the central portion of the filter from the top; and a void forsurrounding the filter between the second pressing part and thirdpressing part.

It is effective that the primary side portion of the filter is exposedoutside at the upper face of the biosensor. It is also effective thatthe secondary side portion of the filter and the working electrode arenot in contact with each other.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an exploded perspective view of a biosensor in accordance withone embodiment of the present invention.

FIG. 2 is a perspective view of a biosensor in accordance with oneembodiment of the present invention.

FIG. 3 is a schematic vertical sectional view of the biosensorillustrated in FIG. 2.

FIG. 4 is an enlarged sectional view of the vicinity of an electrodesystem of a biosensor in accordance with another embodiment of thepresent invention.

FIG. 5 is a diagram showing a response characteristic of a cholesterolsensor in an example of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

As thus described, the present invention relates to a biosensorcomprising: a sample solution supply pathway which includes an electrodesystem and a reagent and has an air aperture on the termination sidethereof; and a filter which is disposed between the sample solutionsupply pathway and a sample supply part and which filters out hemocytes,where plasma with which is obtained by filtering out hemocytes thereinfiltered with in blood by the filter is sucked into the sample solutionsupply pathway due to capillary, and is characterized by furthercomprising: a first pressing part for holding a primary side portion ofthe filter from the bottom; a second pressing part for holding asecondary side portion of the filter from the top and the bottom; athird pressing part for holding the central portion of the filter fromthe top; and a void for surrounding the filter between the secondpressing part and third pressing part. This configuration enablesprevention of destruction of the hemocytes caused by obstructedexpansion of the filter even when a gap between the pressing parts forholding the filter from the top and the bottom is not fitted to thethickness of the filter expanded. Further, dropwise addition of a samplesolution directly onto the filter can inhibit a measurement error thatmay occur as the hemocytes flow along the surface of the filter into thesample solution supply pathway.

In the present description, “a primary side portion” of the filterrefers to a portion closer to the sample supply part for supplying thesample while “a secondary side portion” of the filter refers to aportion closer to the sample solution supply pathway where the electrodesystem is disposed. Further, the base plate side of the filter isreferred to as “the bottom” while the opposite side to the base plate ofthe filter “the top”.

Specifically, at the primary side portion of the filter, the firstpressing part is in contact only with the bottom side of the filter sothat the filter can expand upward when sucking the sample solution.Further, at the central portion of the filter, the third pressing partis in contact only with the top side of the filter so that the filtercan expand downward when sucking the sample solution. Moreover, also atthe void that exists so as to surround the filter between the secondpressing part and the third pressing part, the filter having sucked inthe sample solution can expand. In the absence of the void surroundingthe filter, there is a possibility that the hemocytes, havingtransmitted to the part for pressing the filter without passing throughthe filter, may flow into the electrode system. As thus described,reduction of as many parts that prevent a size change of the filtercaused by the expansion thereof, as possible allows free charge in poresize of the filter as well as filtration free from the destruction ofthe hemocytes.

In addition, at the primary side portion of the filter in the biosensorof the present invention, the first pressing part is in contact onlywith the bottom side of the filter so that an opening can be provided atthe upper part to make the primary side portion exposed outside thebiosensor, whereby the sample solution can be dropped directly onto thefilter.

Furthermore, the third pressing part present on the upper side of thecentral portion of the filter serves like a weir for preventing thesample solution, dropped from the opening, from flowing along thesurface of the upper side of the filter. It is thereby possible toprevent the sample solution from infiltrating into the sample solutionsupply pathway without undergoing filtration process.

The electron mediator for use in the present invention can be selectedfrom potassium ferricyanide or a redox compound having the electrontransferring ability to and from oxidoreductase such as cholesteroloxidase.

Oxidoreductase is an enzyme whose substrate is an object to be measured,and glucose oxidase is applied to a sensor where glucose is the objectto be measured. For measurement of a cholesterol value in blood serum tobe used as a diagnostic indicator, cholesterol oxidase which is anenzyme for catalyzing an oxidation reaction of cholesterol, orcholesterol esterase which is an enzyme for catalyzing the process ofchanging cholesterol dehydrogenase and cholesterol ester to cholesterol,is used. Because the enzyme reaction of cholesterol esterase proceedsvery slowly, with an appropriate surfactant added thereto, activity ofcholesterol esterase can be improved to reduce the time required for theoverall reaction.

These are disposed as a reaction layer on or in the vicinity of theelectrode system in the sensor. These may also be mixed with aconductive material constituting the electrode and a reagent to form theelectrode system. In a sensor which is combined with the base plateprovided with the electrode system and comprises a cover member, whichforms the sample solution supply pathway for a supply of the samplesolution to the electrode system between the base plate and the sensor,these can be provided at the part exposed to the sample solution supplypathway, the opening of the sample solution supply pathway, or the like.Wherever the place is, it is preferable that the sample solutionintroduced can dissolve the reagent with ease and then arrive at theelectrode system. It is preferable to form the hydrophilic polymer layerin contact with the upper face of the electrode system so as to protectthe electrode and prevent the reagent formed from being peeled off.Besides the electrode system, it is also preferable that the hydrophilicpolymer layer is formed as the base of the reagent as formed or it isincluded in the bottom-layer reagent.

It is preferable that the layer including the electron mediator isseparated from the surfactant for enhancing the solubility. It is alsopreferable that it is separated from enzyme cholesterol oxidase andcholesterol esterase, which catalyze the oxidation reaction ofcholesterol, for the sake of preservative stability.

With respect to a biosensor for measuring a blood sugar level, there isan example where a layer containing lipid is formed so as to cover alayer formed on the electrode system, or the like, to facilitateintroduction of the sample solution to the reagent (e.g. JapaneseLaid-Open Patent Publication No. 2-062952). In the biosensor formeasuring cholesterol in accordance with the present invention, it ispreferable to form part of the reagent by freeze-drying (e.g. JapanesePatent Application No. 2000-018834) or to provide hydrophilicity to thesurface of a cover member by processing by means of a surfactant, plasmairradiation or the like. Application of such a configuration caneliminate the need for formation of a lipid layer.

The examples of the hydrophilic polymer to be used include water-solublecellulose derivatives such as, ethyl cellulose, hydroxypropyl celluloseand carboxymethyl cellulose in particular, and polyvinyl pyrrolidone,polyvinyl alcohol, gelatin, agaraose, polyacrylic acid and the saltsthereof, starch and the derivatives thereof, polymers of maleicanhydride or the salts thereof, polyacrylamide, methacrylate resin, andpoly-2-hydroxyethyl methacrylate.

The examples of the surfactants include n-octyl-β-D-thioglucoside,polyethylene glycol monododecyl ether, sodium cholate,dodecyl-β-maltoside, sucrose monlaurate, sodium deoxycholate, sodiumtaurodeoxycholate, N,N-bis (3-D-gluconcamidopropyl) deoxycholcamide andpolyoxyethylene (10) octyl phenyl ether.

As for the lipid favorable used in an amphipathic phospholipid such aslecithin, phosphatidyl choline or phosphatidyl ethanolamine.

As the measuring method for the oxidation current, a two-electrodesystem composed only of a measurement electrode and a counter electrodeand a three-electrode system further comprising a reference electrodeare applicable, and in the three-electrode system, more accuratemeasurement is possible.

In the following, the present invention will be described in detail withthe use of concrete embodiments.

FIG. 1 is an exploded perspective view of a biosensor in accordance witha preferred embodiment.

An insulating base plate 1 is made of an insulating resin e.g.polyethylene terephthalate. On the left side part of the base plate 1 inFIG. 1, an electrode system including a working electrode 2 and acounter electrode 3 is formed by forming a palladium thin film by meansof vapor deposition or sputtering, followed by laser trimming. The areaof the electrode is determined corresponding to a width of a slit 9formed on a spacer 6 laser described. An aperture 4 is formed in thebase plate 1.

A spacer 6 to be combined with the base plate 1 comprises an auxiliaryslit 7 for accommodating a filter 5 therein, a slit 9 constituting asample solution supply pathway 9′, and an opening 8, through which theauxiliary slit 7 communicates with the slit 9.

In a cover 10 formed are an aperture 11 and an air aperture 12; in anauxiliary plate 13 formed are an aperture 14 for supplying a samplesolution to the filter 5, an aperture 15, and a partition portion 18 toserve as a third pressing part.

In an auxiliary upper cover 16 formed is an aperture 17 constituting asample solution supply part for dropping the sample solution onto thefilter 5, and an auxiliary lower cover 20 is composed of a flat plate.In integration of each of the members shown in FIG. 1, the right side ofthe auxiliary slit 7, the right side part of the aperture 11 formed inthe cover 10, the aperture 14 formed in the auxiliary plate 13 and theaperture 17 formed in the auxiliary upper cover 16 in FIG. 1 arecommunicated.

The filter (hemocyte-filtering out portion) 5 is made of glass-fiberfilter paper, and has an isosceles triangle shape with a bottom of 3 mmand a height of 5 mm in the projection thereof drawing to the plane facewhich is the same as the base plate 1. A semicircular portion with aradius of 0.4 mm (not shown) is formed at the tip of the secondary sideportion. The filter 5 has a thickness of about 300 to 400 μm.

For fabrication of this sensor, first, the base plate 1 is placed on anauxiliary lower cover 20 such that the right edge of the auxiliary lowercover 20 is aligned with that of the base plate 1 in such a positionalrelation shown by the dashed line in FIG. 1 to obtain a joint base plateA.

Next, in such a positional relation shown by the dashed line in FIG. 1,the cover 10 and the spacer 6 are combined such that the respectiveright sides thereof, namely the parts corresponding to the bottom sidesof the isosceles triangle shapes formed in the aperture 11 and theauxiliary slit 7 in the projection thereof drawing to the plane facewhich is the same as the base plate 1, are aligned with each other, toobtain a joint base plate B. At that time, a reaction layer is formed,as later described, on the part of the cover 10 facing the slit 9,namely on the upper side part of the sample solution supply pathway 9′.

Furthermore, the joint base plate A comprising the base plate 1 and theauxiliary lower cover 20 and the joint base plate B comprising the cover10 and the spacer 6 are combined in such a positional relation shown bythe dashed line in FIG. 1, and in the projection thereof drawing to theplane face which is the same as the base plate 1, the filter 5 isdisposed such that the right edge (bottom side) of the primary sideportion of the filter 5 having an isosceles triangle shape is alignedwith the right end pairs of the aperture 11 and the auxiliary slit 7. Inother words, the filter 5 is in the state of being disposed on the baseplate 1 while being fitted in the auxiliary slit 7 of the space 6 andthe aperture 11 of the cover 10.

Moreover, the tip of the secondary side portion of the filter 5 getsinto the sample solution supply pathway 9′ formed by the slit 9 and isinterposed between the base plate 1 and the cover 10, and thisinterposed part constitutes a second pressing part. A detaileddescription will be given later. Finally, the auxiliary plate 13 and theauxiliary upper cover 16 are disposed on the cover 10 such that theright ends of the apertures 14 and 17 are aligned with the right ends ofthe aperture 11 of the cover 10 and the auxiliary slit 7 of the spacer 6in such a positional relation shown by the dashed line in FIG. 1.

A schematic perspective view of the biosensor thus obtained is shown inFIG. 2. The cross sectional structure thereof is shown in FIG. 3. FIG. 3is a schematic vertical sectional view of the biosensor of the presentinvention and corresponds to the X—X line cross sectional view shown inFIG. 2. In the biosensor of the present invention shown in FIG. 2, theapertures 15 and 4 for making the filter 5 not in contact with the othermembers are formed, as shown in FIG. 3.

That is to say, as shown in FIG. 3, there are formed: a first pressingpart “a” for holding a primary side portion of the filter 5 from thebottom of the filter 5; a second pressing parts “b” and “b′” for holdinga secondary side portion of the filter 5 from the top and the bottom ofthe filter 5; a third pressing part “c” for holding the central portionof the filter 5 from the top; and an aperture (void) 15 for surroundingthe filter 5 between the second pressing parts “b” and “b′” and thethird pressing part “c”. Further, a cavity exists at the partcorresponding to the part under the filter 5 as well as under the thirdpressing part “c”, which forms the aperture (void) 4 communicating tothe aperture 15.

FIG. 4 is a schematic vertical sectional view showing still another modeof a biosensor of the present invention. A reaction layer and anelectrode system are omitted from FIG. 2, whereas the reaction layer andthe electrode system are shown in FIG. 4. A hydrophilic polymer layer 21and a reaction layer 22a are formed on the electrode system (2 and 3) ofthe base plate 1. Further, a reaction layer 22b is formed on the lowerface side of the cover 10 corresponding to the sealing of the samplesolution supply pathway. It is to be noted that the other members shownin FIG. 4 are equivalent to those shown in FIG. 3.

While the biosensors shown in FIGS. 1 to 4 are produced using six typesof members so as to make the structures thereof easy to understand, theauxiliary upper cover 16 and the auxiliary plate 13, or furtherincluding the spacer 10, may be composed of one member. The lowerauxiliary cover 20 and the base plate 1 may further be composed of onemember.

For measurement of cholesterol in blood with the use of this sensor,blood as the sample solution is supplied from the sample solution supplypart constituted by the aperture 17 of the auxiliary upper cover 16 tothe part (sample supply part) for holding the filter 5. The bloodsupplied here infiltrates from into the upper surface of the primaryside portion of the filter 5 thereinto . In the filter 5, plasma exudesfrom the termination of the secondary side portion of the filter 5because the infiltrating rate of hemocytes is slower than that of theplasma which is a liquid component. The exuded plasma then fills theentire sample solution supply pathway 9′ constituted by the slit 9extended to the vicinity of the electrode system and further to the partof the air aperture 12, while dissolving a reaction layer carried on theposition covering the electrode system and/or the reverse face of thecover. Once the entire sample solution supply pathway 9′ is filled withthe liquid, the flow of the liquid in the filter 5 also stops and hencethe hemocytes are held in the filter 5 at that time, without arriving atthe termination of the secondary side portion of the filter 5. It istherefore necessary to design the filter 5 so as to have a difference inflow resistance between the plasma and the hemocytes to the extent that,when the plasma of enough an amount to fill the entire sample solutionsupply pathway 9′ passes through the filter, the hemocytes do not reachthe secondary side portion of the filter 5. A depth filter with a poresize of about 1 to 7 μm is favorably applied to the filter of thepresent invention. In the case of the example of the present invention,the filter favorably has a thickness of 300 to 400 μm.

After undergoing such a process of filtering out the hemocytes, achemical reaction of the reaction layer dissolved by the plasma with acomponent to be measured (cholesterol in the case of a cholesterolsensor) in the plasma occurs, and a current value in the electrodereaction is measured after a lapse of a certain period of time todetermine a component in the plasma.

FIG. 4 shows an example of disposition of the reaction layer in thevicinity of the electrode system of the sample solution supply pathway9′. On the electrode system of the base plate 1 formed are thehydrophilic polymer layer 21 such as sodium carboxymethyl cellulose(hereinafter simply referred to as “CMC”) as well as the reaction layer22a including a reaction reagent e.g. the electron mediator. Thereaction layer 22b including oxidoreductase is formed on the surfaceexposed to the sample solution supply pathway 9′ on the reverse face ofthe cover member, which is given by combining the cover 10 and thespacer 6.

As shown in FIGS. 1 to 4, the cross sectional area, vertical to thedirection of the flowing liquid, of the sample solution supply pathway9′ constituted by the slit 9 is made smaller than the cross sectionalarea of the primary side portion of the filter 5, however, the part at adistance of 1 mm from the secondary side portion of the filter 5 iscompressed and disposed in the vicinity of the opening 8 of the samplesolution supply pathway 9′. In the case of suction power of a sensorhaving the size described in the example of the present invention, thepart of the filter 5 to be compressed was favorably at a distance of nolonger than about 1 mm from the termination of the secondary sideportion. Further, with respect to the degree of compression of thesecondary side portion of the filter 5, it was preferably that thesecondary side portion was compressed into about one fourth to one thirdof the primary side portion. While it is difficult to represent thesuction power to the sensor by a numeric value in such a compressingcondition, in the case of a spacer with a thickness of 100 μm, a filterwith a thickness of 370 μm exhibited a favorable measurement result(flow-in rate). It should be noted that, in the case of the filter witha thickness of 310 μm or less, the flow-in rate was slower.

As thus described, making the cross sectional area of the samplesolution supply pathway 9′ smaller than the cross sectional area of theprimary side portion of the filter 5 allows rapid suction of plasma,with which is obtained by filtering out hemocytes therein filtered within blood by the filter 5, into the sample solution supply pathway 9′ dueto capillarity.

The reaction layer generally comprises an easy-to-dissolve part and ahard-to-dissolve part. A portion of the reaction layer at the edge ofthe sample solution supply pathway 9′, i.e. the part along the wall faceof the slit 9 in the spacer 6 is easy to dissolve, whereas the centralportion of the reaction layer in the flowering direction of the liquidis hard to dissolve. Since the sample solution having passed through thefilter 5 flows along the spacer 6 by priority, there may be cases whenthe sample solution fills in the air aperture before completedissolution of the central portion of the reaction layer. Protrusion ofthe central portion of the secondary side portion of the filter 5 intothe sample solution supply pathway 9′ more than the both the right andleft terminations thereof enables the priority flow of the samplesolution through the central portion of the sample solution supplypathway 9′, whereby the plasma can be rapidly flown into the senorwithout leaving bubbles at the central portion of the sample solutionsupply pathway 9′.

In-measurement, when blood as the sample solution is supplied from thesample solution supply part constituted by the aperture 17 of theauxiliary upper cover 16 to the filter 5, the blood infiltrates from theupper surface of the primary side portion of the filter 5 thereinto. Inthe presence of the third pressing part “c” to serve as a partition atthat time, dropwise addition of the sample solution onto the surface ofthe filter 5 will not be followed by priority flowing of the samplesolution along the surface of the filter 5 directly into the samplesolution supply pathway 9. Further, in the projection thereof drawing tothe plane face which is the same as the base plate 1, the position ofthe third pressing part “c” does not correspond to that of a firstpressing part “a”, whereby neither the expansion of the filter 5 isobstructed nor there is the fear of destroying the hemocytes.

It is preferable that the electrode system comprises a noble metalelectrode. With the width of the sample solution supply pathway beingpreferably not more than 1.5 mm, accuracy in determination of anelectrode area is poor in a printing electrode processed by screenprinting. As opposed to this, the noble metal electrode exhibits a highaccuracy in determination of the electrode area as being able to besubjected to laser trimming by a width of 0.1 mm.

Below, an example of the present invention will be described; however,the present invention is not limited to this.

EXAMPLE

A cholesterol sensor having the configurations of FIGS. 1 to 4, wherethe reaction layer 22a included the electron mediator and the reactionlayer 22b included cholesterol oxidase, cholesterol esterase and asurfactant, was produced.

First, 5 μl of an aqueous solution containing 0.5 wt % of CMC wasdropped onto the electrode system of the base plate 1, and dried in adrying apparatus with warm blast at 50° C. for 10 minutes to form theCMC layer 21.

Next, 4 μl of potassium ferricyanide aqueous solution (corresponding to70 mM of potassium ferricyanide) was dropped onto the CMC layer 21, anddried in the drying apparatus with warm blast at 50° C. for 10 minutesto form the layer 18a including potassium ferricyanide.

Polyoxyethylene (10) octyl phenyl ether (Triton X100) as the surfactantwas added to an aqueous solution with cholesterol oxidase originatingfrom Nocardia (EC1.1.3.6) and cholesterol esterase originating fromPseudomonas (EC3.1.1.13) dissolved therein. 0.64 μl of this mixedsolution was dropped onto the part (sample supply pathway 9′) of theslit 9 formed by integrating the cover 10 with the spacer 6, prefrozenwith liquid nitrogen at −196° C., and dried in a freeze-drying apparatusfor two hours, to form the reaction layer 22b including 570 U/ml ofcholesterol oxidase, 1,425 U/ml of cholesterol esterase, and 2 wt % ofthe surfactant.

The slit 9 had a width of 0.08 mm and a length (the length between theopening of the sample solution supply pathway 9′ and the air aperture)of 4.5 mm. The spacer 6 had a thickness (the distance between the baseplate 1 and the cover 10) of 100 μm.

As for the filter 5 used is one made of a glass fiber filter having athickness of about 370 μm in an isosceles triangle shape with a bottomof 3 mm and a height of 5 mm. The tip of the secondary side portion (thepart in contact with the opening 8 of the sample solution supply pathway9′) was roundly processed and then placed between the joint base plate Acomprising the base plate 1 and the auxiliary lower cover 20 and thejoint base plate B comprising the cover 10 and the spacer 6.

Subsequently, the member obtained by placing the filter 5 between thejoint base plate A and the joint base plate B was bonded to the memberobtained by integrating the auxiliary plate 13 with the auxiliary uppercover 16, to produce a cholesterol sensor having the structures shown inFIGS. 1, 2 and 4.

10 μl of whole blood as the sample solution was introduced into thesample solution supply part of this sensor; three minutes later, a pulsevoltage of +0.2 V was applied to the measuring electrode toward theanode relative to the counter electrode, and five second later, a valueof a current flowing between the working electrode and the counterelectrode was measured. The results were shown in FIG. 5. FIG. 5 is agraph showing relations between the total cholesterol concentration andthe response current.

As is evident from FIG. 5, according to the sensor of the presentinvention, a favorable linearity between the cholesterol concentrationand the response current value was obtained. In FIG. 5, “x” indicatesthe result of plasma with a ratio of red cell volume of 0%, “O”indicates the result of whole blood with a ratio of red cell volume of35%, and “□” indicates the result of whole blood with a ratio of redcell volume of 60%.

Industrial Applicability

According to the present invention, hemocytes as interfering substancescan be removed without the destruction thereof by a filter, and plasmawith which is obtained by filtering out hemocytes therein removed inblood can be supplied with rapidity to an electrode system even with thethickness of the filter being thin. Accordingly, there can be provided achemical biosensor excellent in response characteristic.

1. A biosensor comprising: Anan insulating base plate; an electrodesystem having a working electrode and a counter electrode which areprovided on said base plate; a reagent including at least oxidoreductaseand an electron mediator; a sample solution supply pathway whichincludes said electrode system and said reagent and has an air apertureon the termination side thereof; a sample supply part; and a filterwhich is disposed between said sample solution supply pathway and saidsample supply part and which filters out hemocytes, where plasma withhemocytes therein filtered with said filter is sucked into said samplesolution supply pathway due to capillarity , characterized by furthercomprising: a first pressing part for holding a primary side portion ofsaid filter from the bottom; a second pressing port part for holding asecondary side portion of said filter from the top and the bottom; athird pressing part for holding the central portion of said filter fromthe top; and a void for surrounding said filter between said secondpressing part and third pressing part.
 2. The biosensor in accordancewith claim 1, characterized in that said primary side portion of saidfilter is exposed outside at the upper face of the biosensor.
 3. Thebiosensor in accordance with claim 1, characterized in that saidsecondary side portion of said filter and said working electrode are notin contact with each other.
 4. The biosensor in accordance with claim 2,characterized in that said secondary side portion of said filter andsaid working electrode are not in contact with each other.